1. Field of The Invention
The present invention relates to a method for determining antistreptolysin O antibody. More particularly, the present invention is concerned with a method for immunologically determining antistreptolysin O antibody, which comprises contacting a test sample solution containing antistreptolysin O antibody with a binding agent capable of specifically binding the antistreptolysin O antibody thereto, thereby selectively binding the antistreptolysin O antibody to the binding agent by antigen-antibody reaction, and determining an amount of the antistreptolysin O antibody bound to the binding agent, wherein the binding agent comprises a streptolysin O-immobilized carrier obtained by contacting a solution containing streptolysin O with a carrier having immobilized thereon at least one specific steroid. The binding agent to be used in the method of the present invention is capable of specifically binding the antistreptolysin O antibody thereto, so that the determination of antistreptolysin O antibody can be readily conducted with high specificity and accuracy. Further, the method of the present invention need not use erythrocytes, which are unstable and have different qualities depending on the lot of erythrocytes. Due to the above-mentioned advantages, the method of the present invention enables automation of specific and accurate determination of the antistreptolysin O antibody, differing from conventional determination methods which cannot be employed to automate specific or accurate determination of the antistreptolysin O antibody. The method of the present invention can be advantageously used for the diagnosis of infection with group A hemolytic streptococci.
2. Description of Related Art
Antistreptolysin O antibody is an antibody against streptolysin O. Streptolysin O is a toxin (which is a member of the group of thiol-activated bacterial cytolysins) produced, for example, by group A hemolytic streptococci classified according to the serological classification by Lancefield (see Lancefield, R. C., J. Exp. Med., Vol. 57, pp.571-595, 1933), and is generally known as a protein having a molecular weight in the range of from 50,000 to 70,000. Streptolysin O is known to interact with cholesterol and related sterols (see Prigent, D. et al., BBA, Vol. 443, pp.288-300, 1976), and exert cytolytic effects on a broad range of mammalian cells. Streptolysin O lyses erythrocytes, usually under reductive conditions, e.g. in the presence of mercaptoethanol, by an interaction with membrane cholesterol. It is known that antistreptolysin O antibody level is elevated in the serum from a patient infected with group A hemolytic streptococci.
Group A hemolytic streptococci are pathogenic microorganisms which are causative of various diseases including tonsillitis, pharyngitis, skin purulence and scarlet fever, and secondary diseases, such as rheumatic fever, glomerulonephritis, and the like. Most of these diseases manifest no specific clinical symptoms from which pathogenic microorganisms causing the diseases can be identified and, therefore, for clinical diagnosis of infections with group A hemolytic streptococci, confirmation of the presence of antistreptolysin O antibody and determination of the antibody titer have generally been utilized see Tomika Nagata, "Rinshokensa (Laboratory Examinations)", Vol.23, Supplementary Edition, pp.1172-1175, 1979!.
As methods for the determination of antistreptolysin O titer, the Rantz-Randall method using rabbit erythrocytes, sheep erythrocytes or human type O erythrocytes (see L. A. Rantz et al., Proc. Soc. Exp. Biol. Med., Vol.59, pp.22-25, 1945), and a modification of the Rantz-Randall method, i.e., the microtiter method (see Edwards, E. A.; J. Bacteriol., Vol.87, pp.1254-1255, 1964) have conventionally been widely used in routine assays. Both determination methods are based on the principle that antistreptolysin O antibody in a test sample solution neutralizes the hemolytic activity of streptolysin O.
Group A hemolytic streptococci, which have infected living bodies, produce not only streptolysin O, but also various other antigens, such as erythrogenic toxin, streptokinase, streptodornase, hyaluronidase, ribonuclease and neuraminidase and, in addition, various antibodies against these antigens occur in the blood see "Rensakyukin-kansensho II: Sono Kiso-to-Rinsho (Streptococcicosis, Volume II: the basic and clinical)"; edited by Yuichi Shiokawa, Morimasa Yoshioka and Shigeyuki Hamada; pp.317-359, Hirokawa Shoten K. K., Japan, Jun. 25, 1992!. However, since the above determination methods utilize the hemolytic activity characteristic of streptolysin O, streptolysin O to be used as a reagent for the above determination methods need not necessarily be a purified one, and in the above determination methods, non-purified streptolysin O can be used for specifically recognizing and determining only antibody against streptolysin O, i.e. antistreptolysin O antibody, among antibodies against a number of antigens Fujimoto et al., "Rinsho-byori (Clinical Pathology)", Vol.40, pp.21-27, 1992!.
However, these determination methods require the use of fresh erythrocytes. Erythrocytes are unstable and different in quality between different lots, so that these determination methods using erythrocytes are likely to be unstable. Further, these determination methods necessarily involve complicated operations. Therefore, it is very difficult to automate these determination methods (see European Patent Application Publication No. 0 475 786 A2 corresponding to Unexamined Japanese Patent Application Laid-Open Specification No. Hei 6-186233).
In recent years, a determination method for antistreptolysin O antibody titer which is based on the principle of immunoagglutination and uses a carrier (such as colloidal latex particles) having streptolysin O immobilized thereon, has been used see T. Miura et al., "Eisei-kensa (Hygienic Examinations)", Vol.36, pp.36-40, 1987!. This conventional determination method using a streptolysin O-immobilized carrier is improved in that unstable erythrocytes need not be used and that this method can be automated, thus differing from the above-mentioned conventional determination methods utilizing as a criterion the hemolytic activity of streptolysin O.
However, the conventional determination method using a streptolysin O-immobilized carrier has the following disadvantages. Streptolysin O is generally prepared from a culture mixture of group A hemolytic streptococci. It is very difficult to obtain a highly purified streptolysin O from such a culture mixture, so that streptolysin O obtained from such a culture mixture of group A hemolytic streptococci usually contains a wide variety of other antigens produced by group A hemolytic streptococci. Therefore, when such streptolysin O is immobilized on a carrier, other antigens produced by group A hemolytic streptococci are also immobilized on the carrier together with the streptolysin O. Thus, the antibody titer which can be obtained by the conventional determination method using the above-mentioned streptolysin O-immobilized carrier is one which is obtained with respect to all antibodies which react with the various antigens immobilized on the carrier. Therefore, this conventional determination method using a streptolysin O-immobilized carrier has a problem in that it is unsatisfactory in reaction specificity to antistreptolysin O antibody see Fujimoto et al., "Rinsho-byori (Clinical Pathology)", Vol.40, pp.21-27, 1992!.
In these situations, it has been desired to develop a determination method which solves the problems accompanying the conventional methods for the determination of antistreptolysin O antibody. That is, it has been desired to develop a method for the determination of antistreptolysin O antibody, which can be used for accurately, easily determining antistreptolysin O antibody with high reaction specificity, and which can be performed by automated operation as well as by manual operation.